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5-Carboxyfluorescein is a single isomer derivative of 5(6)-carboxyfluorescein (Item No. 17172) that can be used to fluorescently label biomolecules through the interaction of carboxylic acid with primary amines. It demonstrates excitation/emission maxima of 492 and 518 nm, respectively.

Orange Solid

5-Carboxyfluorescein is a single isomer derivative of 5(6)-carboxyfluorescein that can be used to fluorescently label biomolecules through the interaction of carboxylic acid with primary amines. It demonstrates excitation/emission maxima of 492 and 518 nm, respectively.

A useful reagent for the preparation of hydrolytically stable fluorescent conjugates and is a useful starting material for the synthesis of other fluorescein-derives reagent

Derivative of fluorescein giving stable derivative upon conjugation with biopolymers.

5-Carboxyfluorescein (5-FAM) is one of the most widely used fluorescent dyes. Its excitation spectrum peak (EXmax) is at 492 nm, whilst its fluorescence emission spectrum peak (EMmax) is at 518 nm; it possesses a high quantum yield and is used for labelling oligonucleotides [2]. 5-FAM is a green fluorescent compound containing a single carboxyl group, which can be used as a detection substrate for OAT1 and OAT3. Its fluorescence intensity is extremely sensitive to pH. In acidic conditions (pH < 6), its fluorescence is significantly quenched; it is suitable for use in weakly alkaline buffers with a pH of 7.5–9.0.

ChEBI: 5-carboxyfluorescein is a monocarboxylic acid. It has a role as a fluorochrome. It is functionally related to a fluorescein (lactone form).

reagent type: fluorescent-labelling reagent

5-Carboxyfluorescein is used for the staining of cells to differentiate between live and dead cells. It helps in monitoring the integrity and intactness of the cell membrane. It is also used for the measurement of intracellular pH. 5-Carboxyfluorescein diacetate is a non-fluorescent molecule which is cleaved by intracellular esterases, thereby giving a green fluorescent product 5-carboxyfluorescein. In viable cells, the fluorescent molecule is impermeable to the cellular membrane.

5-Carboxyfluorescein is a mixture of 5- and 6-isomers prepared by the reaction of benzotricarboxylic anhydride with resorcinol and zinc chloride.
Reaction: 1,2,4-Benzenetricarboxylic anhydride (also called 4-carboxyphthalic anhydride, 25.0 g, 0.13 mol) was added to a solution of 1,3-dihydroxybenzene (also called resorcinol, 28.6 g, 0.26 mol) in methane sulfonic acid (1M). An air condenser was attached to the flask and the reaction was heated at 85°C in an open vessel for 24 h. After cooling to room temperature, the reaction mixture was poured into 7 volumes of ice/water. An orange-yellow precipitate formed; this was collected by filtration and dried in an oven at 200°C. This residue was recrystallized two times from methanol/hexane to give 1.0 g of 6-carboxyfluorescein methanesulfonic acid adduct 2b. The mother liquors from this procedure were collected, the solvent was removed in vacuo, and the residues were recrystallized two times from ethanol/hexanes to give 3.2 g of 5-carboxyfluorescein methanesulfonic acid adduct 2a.

The methane sulfonic esters 2 are easily converted to the 5- and 6-carboxyfluoresceins 1 by treatment with sodium hydroxide solution then neutralizing with aqueous HCl.

[1] RAINER FISCHER. Extending the Applicability of Carboxyfluorescein in Solid-Phase Synthesis[J]. Bioconjugate Chemistry Bioconjugate, 2003, 14 3: 653-660. DOI: 10.1021/bc025658b